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K. Sayanagi, Y. Li, M. E. Rayborn, K. G. Shadrach, X. Yang, V. Perez, R. G. Salomon, J. G. Hollyfield; Electron Microscopic Analysis of Drusen Deposits That Develop Following Immunization With CEP-MSA. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3967.
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Carboxyethylpyrrole (CEP) is a unique protein adduct generated from oxidative damage to docosahexanoic acid (DHA) and is a link between oxidative damage and inflammation in this mouse model for age-related macular degeneration (AMD). To understand the timing and pattern of development of drusen in mice immunized with CEP in mouse serum albumin (MSA), we conducted electron microscopic analysis on the outer retina.
Using standard mouse immunization protocols, mice were anesthetized with ketamine/xylazine in PBS (80-90 mg/Kg ketamine, 2-10 mg/ml xylazine). The mice were then immunized with 200µg of CEP-MSA in complete Freund’s adjuvant (CFA) in footpads and tail base. Control mice used for comparisons with CEP-MSA were injected only with CFA along with naive and Rag-/- mice. After 12-14 months, the mice were sacrificed and processed for analysis. For electron microscopy, eyes were fixed in 2% glutaraldehyde, 2% formaldehyde in 0.1 M cacodylate buffer overnight and post-fixed in 1% osmium tetroxide, dehydrated in graded ethanol, to propylene oxide and then transferred to a plastic resin mixture containing Polybed 812 and Araldite 502 with polymizer. After polymerization, selected blocks were thin sectioned on an RMC, MT-XL ultramicrotome, placed on nickel grids, stained with uranyl acetate and lead citrate and viewed in a Tecnai 20, 200 kV digital electron microscope equipped with a Gatan image filter. Sub-RPE deposits were measured using an image processing algorithm that recognizes the basal infoldings/sub-RPE deposits in digitized images.
Control mice at 12-14 months have normal basal infoldings; immunized mice show flocculent accumulation of material between the basal lamina, electron dense membranes and a 2-3 fold increase in the thickness of Bruch’s membrane was evident.Electron dense granular material was occasionally present in Bruch’s membrane and evidence of cellular invasion of this usually cell-free lamina was also seen.
A single immunization of CEP-MSA is sufficient to cause drusen formation in basal lamina deposits below the retinal pigment epithelium. We hypothesize that this immunization raises CEP production in the RPE, which stimulates these cells to elaborate this basal lamina debris.
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